Since Martinus Willem Beijerinck proposed the word “virus” for the first time in 1899, more than 6,000 viruses have been identified over the world. Among them, human immunodeficiency virus, hepacivirus, influenza virus, severe acute respiratory syndrome (SARS) virus, enterovirus EV71, and Ebola virus which outbreaks in West African countries with a high lethality, have become natural enemies to human health. Viruses can cause an immense damage even in the early stage of epidemics, including personal injury, social panic, and economic fluctuation, etc., and therefore have a serious impact on the national economy and social stability. In this case, even drugs with a weak therapeutic effect will have an inestimable effect on reducing personal injury, especially eliminating social panic and stabilizing economic fluctuation. The optimum option for reducing the hazards of this new public health emergency is to develop broad-spectrum antiviral agents.
Virus reproduction will not happen in the absence of host cells. Entry of most of viruses into host cells occur mainly via Clathrin-mediated endocytosis (CME), Caveolin-mediated endocytosis, Clathrin and Caveolin-independent endocytosis and Macropinocytosis. Entry of a lot of viruses such as enterovirus EV71, Dengue virus, and Hepatitis C virus into cells occurs mainly via CME. CME is a potential target for broad-spectrum anti-virus in recent years, since its function and mechanism are independent of viruses themselves, and drug resistance does not occur easily with virus variation.
In CME, clathrin, adaptor protein (AP-2), dynamin, and the like, are essential key molecules. The mechanism concerning entry of a virus into a cell via Clathrin-mediated endocytosis has been well studied, and the whole endocytosis process can be divided into the following four stages generally.
1) Nucleation of clathrin-coated pits: in this stage, it is dependent on the participation of a protein complex of FCHo1/2, eps15 and intersectin-1. This complex assembles at coated pit nuclear sites prior to clathrin coat formation and recruits the AP-2 adaptor protein via the multiple AP-2 binding sites of eps15. Clathrin is then recruited to the surface of cytoplasmic membrane by AP-2, and the increased local clathrin concentration exceeds the threshold required for polymerization of the clathrin triskelion, which allows for local assembly of clathrin coat. Clathrin is necessary in stabilizing the shape of coated pits and driving membrane invagination.
2) Cargo capture in coated pits: a virus as cargo is bound to a specific receptor that recognizes the virus on the surface of cell membrane and therefore is captured and detained. The formation of the clathrin network activates Adaptin-associated kinase 1 (AAK1), which phosphorylates the μ2-subunit of AP-2 thereby AP-2 is bound more strongly to phosphatidylinositol diphosphate (PIP2) of membrane to expose the μ2 cargo-binding site. The cargo-AP-2 complex simulates the activity of can activate PIP kinase type Iγ (PIPK Iγ), and contributes to the increase in the PIP2 concentration of coated membrane zone, thus contributes to appearance of sufficient sites to which AP-2, epsin and other CME-associated proteins are bound, which maintains the growth of clathrin-coated pits (CCPs).
3) Curvature induction and membrane invagination: the initial membrane deformation may occur during clathrin-coated pit (CCP) nucleation, where FCHo proteins make a small curvature of membrane through their F-BAR domains. The occurrence of this curvature is putatively mediated through coating of multiple membrane-binding proteins and clathrin, including epsin and amphiphysin, etc. The membrane curvature around the coat forms a growing bud on the cell membrane surface. As the membrane invaginates and buds into a vesicle, a tubular neck region begins to form on the membrane surface, this region has not free PIP2 coated by and contained in clathrin, but has still a high curvature, which attracts proteins containing curvature-sensing N-BAR domains.
4) Vesicle scission and uncoating: once clathrin-coated vesicles have a sufficiently high curvature, dynamin is capable of forming a ring structure around the vesicle neck, and an inward compression force is generated by GTP hydrolysis-triggering mechanism, and vesicle scission is carried out with the participation of proteins such as amphiphysin, and the scissored vesicles are uncoated immediately. The uncoating mechanism may be as follows, in the initial stage, endophilin recruits the synaptojanin with phosphatase activity to the vesicles, thereby disrupting the high affinity between AP-2 and PIP2, resulting in the conversion of PIP2 to PI(4)P. GAK/auxilin bind the newly formed PI(4)P via a PTEN-like domain and recruit the Hsc70 chaperone to the coated vesicle to interact with AP-2 and clathrin, thus the uncoating process is completed. The uncoated vesicles are polymerized or fused to endosomes directly, followed by fusion to lysosomes, the contents are degraded, membrane receptors are separated from virus ligands, and the entire entry of a virus into a cell is finished.
Therefore, the inhibition of clathrin may function to block the endocytosis of a virus. The purpose of the present invention is to synthesize new clathrin inhibitors for use in the preparation of broad-spectrum antiviral agents for preventing and/or treating viral diseases caused by various viruses.